Marina Quiroga

Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives.

Diarrheagenics Escherichia coli are the major agents involved in diarrheal disease in developing countries. The aim of this study was to evaluate the time of appearance of the first asymptomatic infection by the different categories of diarrheagenic E. coli in 44 children since their birth and during the first 20 months of their lives. In all of the children studied, we detected at least one category of diarrheagenic E. coli through the 20 months of the study. 510 diarrheagenic E. coli (33.5%) were obtained from the 1,524 samples collected from the 44 children during the time of the study (31.4% EAggEC, 28.8% EPEC, 27.1% DAEC, and 12.7% ETEC). Neither EHEC nor EIEC were identified. The median age for diarrheagenic E. coli colonization was 7.5 months. The mean weaning period was 12.8 months and the mean age for introduction of mixed feeding (breast fed supplemented) was 3.8 months. A significantly lower incidence of diarrheal disease and asymptomatic infections was recorded among the exclusively breast-fed rather than in the supplemented and non breast-fed infants. For ETEC, EPEC and EAggEC the introduction of weaning foods and complete termination of breast-feeding were associated with an increase of asymptomatic infections.

Antibiotic susceptibility patterns and prevalence of group B Streptococcus isolated from pregnant women in Misiones, Argentina

This study was performed to determine the susceptibility patterns and the colonization rate of Group B Streptococcus (GBS) in a population of pregnant women. From January 2004 to December 2006, vaginal-rectal swabs were obtained from 1105 women attending Dr. Ramón Madariaga Hospital, in Posadas, Misiones, Argentina. The carriage rate of GBS among pregnant women was 7.6%. A total of 62 GBS strains were randomly selected for in vitro susceptibility testing to penicillin G, ampicillin, tetracycline, levofloxacin, gatifloxacin, ciprofloxacin, quinupristin-dalfopristin, linezolid, vancomycin, rifampicin, trimethoprim- sulfametoxazol, nitrofurantoin, gentamicin, clindamycin and erythromycin, and determination of resistance phenotypes. No resistance to penicillin, ampicillin, quinupristin-dalfopristin, linezolid, and vancomycin was found. Of the isolates examined 96.8%, 98.3%, 46.8%, and 29.0% were susceptible to rifampicin, nitrofurantoin, trimethoprim-sulfametoxazol and tetracycline, respectively. Rank order of susceptibility for the quinolones was: gatifloxacin (98.4%) > levofloxacin (93.5%) > ciprofloxacin (64.5%). The rate of resistance to erythromycin (9.7%) was higher than that of other reports from Argentina. High-level resistance to gentamicin was not detected in any of the isolates. Based on our finding of 50% of GBS isolates with MIC to gentamicin equal o lower than 8 μg/ml, a concentration used in one of the selective media recommended for GBS isolation, we suggested, at least in our population, the use of nalidixic acid and colistin in selective media with the aim to improve the sensitivity of screening cultures for GBS carriage in women.

Prospective study of enteropathogens in two communities of Misiones, Argentina

Children under five years of age, from two communities of different socio-economic strata (97 from Zaiman and 55 from Las Dolores) were examined epidemiologically during 2 years, by means of quarterly visits of the working team, who carried out the collection of faecal samples. During the study, one or more enteropathogens were identified in 73.9% of samples in children from Zaiman and in 58.3% of the samples from Las Dolores, being associated to diarrhoea in 70.5% and to asymptomatic infections in 65.7%. The number of diarrheic episodes was higher in Zaiman (15.45%) than in Las Dolores (12.35%), being more frequent in the spring-summer seasons. In Zaiman, the bacterial enteropathogen proportion was relevantly higher (p < 0.005) in children with diarrhoea, whereas the presence of parasites was more frequent in asymptomatic children (p < 0.01). Rotavirus had an even distribution within diarrheic and asymptomatic children. In Las Dolores, no relevant differences were found in the detection of enteroparasites between diarrheic and asymptomatic children. Mixed infections were detected; enterotoxigenic Escherichia coli (ETEC)-rotavirus and ETEC-parasites being the most frequent ones. ETEC was involved in 85% of these infections. These data, together with the high enteropathogen carriage, suggest an elevated level of environmental contamination. The latter plays an important role in diarrheic diseases, and added to the most extreme poverty, it affects childrens lives.

Phenotypic and genotypic characterization of Streptococcus agalactiae in pregnant women: first study in a province of Argentina

Group B Streptococcus (GBS) is the leading cause of neonatal infections. Our purpose was to characterize GBS colonization in pregnant women, current serotypes, resistance phenotypes and genes associated with virulence. In Misiones, Argentina, there are no previous data on this topic. Vaginal-rectal swabs from 3125 pregnant women were studied between 2004 and 2010. GBS strains were identified by conventional and serological methods (Phadebact Strep B Test, ETC International, Bactus AB, Sweden). Serotypes were detected using Strep-B Latex (Statens Serum Institut, Denmark). Resistance phenotypes were determined by the double-disk test. Genes were studied by PCR. Maternal colonization was 9.38%. Resistance to erythromycin was 11.6%, and the constitutive phenotype was the predominant one. Serotype Ia was the most frequent, whereas serotypes IV, VI, VII and VIII were not detected. The lmb, bca and hylB genes were detected in more than 79% of the strains. In this study, the colonization rate with GBS and the serotype distribution were compared with studies reported in other areas of the country. The high resistance to erythromycin in Misiones justifies performing antibiotic susceptibility testing. The serotype distribution, the genes encoding putative virulence factors, and the patterns of resistance phenotypes of GBS may vary in different areas. They thus need to be evaluated in each place to devise strategies for prevention.

Susceptibilities to carbapenems and presence of cphA gene on food-borne Aeromonas

ABSTRACT The purpose of this study was to determine the susceptibilities of food-borne Aeromonas to carbapenems, as well as to investigate the presence of a metallo carbapenemase-encoding gene, named cphA. Minimum Inhibitory Concentration (MIC) was determined following NCCLS standards. All the tested microorganisms were susceptible to imipenem, meropenem and biapenem. However, a strong inoculum size effect on carbapenem MICs was observed for most of the strains. Six strains, out of seven, showed the presence of metallo--β−lactamases but cphA gene was detected in only two strains of A. veronii bv. sobria . Key words: metallocarbapenemase, cphA gene, food borne Aeromonas . * Author for correspondence INTRODUCTION Evolution of bacterial resistance to antibiotics in humans, animals and the environment is the result of the interaction between the exposure to antibiotics, selection of microorganisms carrying primordial genes of resistance, and transmission of resistance genes between bacteria. Selective effects occur in selective compartments, where particular antibiotic concentrations result in a differential growth rate of resistant bacterial variants (Baquero et al., 1998) This may happen even at very low antibiotic concentrations able to select low-level-resistant bacteria. Analysis of selective environment-related antibiotic-host-bacteria interactions is essential to understand the biology of antibiotic resistance. (Baquero et al., 1998) To anticipate emergence of resistance, it is necessary to better understand the genetics and biochemistry of resistance mechanisms and to develop methodologies to foresee their evolution at the individual or population level (Baquero et al., 1998). Most retrospective and prospective studies show that after the introduction of an antibiotic, the level of resistance increases both among pathogenic bacteria and in commensal bacteria (van den Bogaard and Stobberingh, 2000). Moreover, commensal bacteria constitute a reservoir of resistance genes for (potentially) pathogenic bacteria. Their level of resistance is considered to be a good indicator for selection pressure by antibiotic use and for resistance problems to be expected in pathogens. Aeromonas strains have been found to be able to produce up to three different β-lactamases

β-lactam Resistance in Variants of Aeromonas spp. Selected In Vitro Under Antibiotic pressure

During recent decades the role of Aeromonas spp. in a variety of human illnesses has been documented 1. Aeromonas spp. produce several chromosomally mediated inducible b-lactamases, one of them a metallob-lactamase2. The most important clinical aspect of inducible b-lactamase production is the emergence of resistant mutants, with genetically derepressed synthesis of the enzymes, which are associated with therapeutic failures 3. We report here about beta-lactam-resistant variants of Aeromonas spp. carrying the metallo-b-lactamasecphA-encoding gene which were selected in vitro under antibiotic pressure. Twenty-one clinical isolates of Aeromonas spp. were included in the present study. The strains were isolated from patients with extraintestinal infections by Aeromonas spp. (19 skin, soft tissue and bone infections, 1 abdominal infection and 1 lung abscess) diagnosed in Hospital Dr. Ramón Madariaga and private clinics of Posadas, Misiones, Argentina from January 2002 to December 2006. All isolates (19 A. hydrophila, 1 A. jandaei and 1 Aeromonas spp.) were identified using conventional techniques 4. To determine the presence of pre-existing mutants in the initial population, an overnight blood agar culture was removed and inoculated onto tripticase soy (Ts) broth. The inoculum was adjusted to 108-109 CFU/ml and diluted 1/10 and 1/100 in Ts. One hundred (100) μl of each bacterial suspension were inoculated onto blood agar containing a concentration of antibiotic equivalent to 10 ́ minimum inhibitory concentration (MiC) of cefotaxime (CTx) of each strain. The numbers of colonies obtained after 18 h incubation at 35°C were recorded. The selection of stably derepressed mutants was studied as follows, 50 μl of inocula of 108-9 CFU/ml were incubated for 18 h at 35°C in Mueller Hinton (MH) broth containing 0.5 MiC of CTx of each strain. An aliquot of 50 μl of each suspension was removed and inoculated onto fresh MH broth containing the CTx MiC of each strain and incubated for another 18 h at 35°C. The procedure was repeated with MH broth containing successively 2 ́MiC and 10 ́MiC of the antibiotic. Finally, an aliquot of the bacterial suspension was removed and inoculated onto blood agar without antibiotic. The microorganisms were subcultured seven times in antibiotic-free MH broth. The susceptibility to imipenem (iMP) was tested by broth microdilution method 9 after the last passage. We considered as selection of stably derepressed mutants the growth of organisms that demonstrated at least a 4-fold increase in the iMP MiC when compared with their parent strains and remained stable when the MiC was repeated. The MiCs of ampicillin-sulbactam (Laboratorios Bagó, Argentina), cephalothin (Argentia s.A.C.i.F.i., Argentina), ceftazidime (GlaxosmithKline, Argentina), CTx (Argentia s.A.C.i.F.i., Argentina), cefepime (Bristol-Myers squibb, Argentina) and iMP (Merck sharp & Dohme, Argentina) of the 21 Aeromonas spp. strains and the derepressed mutants were determined using broth microdilution methods as described previously 5. Breakpoint interpretive criteria used were those established by the CLsi 6. The quality control strains used were escherichia coli ATCC 25922, escherichia coli ATCC 35218 and pseudomonas aeruginosa ATCC 27853. The screening tests for phenotypic detection of metallo-b-lactamases were performed without knowledge of the genetic results. The 21 Aeromonas spp. strains and the derepressed mutants were checked for production of metallo-b-lactamase by the modified Hodge test 7, the iMP-EDTA disk method 8 and iMP-EDTA disk synergy test 7. To confirm the phenotypic results the MiC of iMP plus EDTA was done as described by Migliavacca et al. 9. PCR analysis to detect the presence of the cphA gene was performed as described by Massidda et al.10. Chromosomal DnA was extracted from the 21 Aeromonas spp. strains and the derepressed mutants by boiling cell suspensions for 10 min. The primers used were Ahy-ssD/F (5’ GCT TAG AGC TCC TAA GGA GCA AGA TGA AAG GTT GG 3’) and AhyKpn/R (5’ GCA TAG GTA CCT TAT GAC TGG GGT GCG GCC TTG 3’) 10. Pre-existing derepressed mutants were not isolated in this study. Of the 21 Aeromonas strains under antibiotic pressure, only 6 stably derepressed mutants were selected from the serial passage in increasing concentrations of CTx. REpRInt